SARS-CoV-2 rapidly evolves lineage-specific phenotypic differences when passaged repeatedly in immune-naïve mice

The persistence of SARS-CoV-2 despite the development of vaccines and a degree of herd immunity is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of wild-type (WT) SARS-CoV-2 in Balb/c mice yield mouse-adapted strains with greater infectivity and mortality. We investigate if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing the human ACE2 receptor, in a BSL-3 laboratory without selective pressures, drives human health-relevant evolution and if evolution is lineage-dependent. Late-passage virus causes more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurs with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, is co-inherited at times with spike E1182G per Nanopore sequencing, existing in different within-sample viral variants at others. Both S371F and E1182G are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2’s tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribution.

7) Why in Figure 3 authors have now two different Delta P20 viruses?What lineage do they use to perform the experiments described in Figure 2? 8) Images in Figure 4 are very small to appreciate the details. 9) In this sentence: "On day 3 post infection, Beta P20 virus induced greater production of proinflammatory CXCL1, CCL2 and IL-6 versus P0 (Figures 6A-C)", the reference to the Figure is incorrect, I think it shoud refer to Figure 6C,D,E.Also, apply to the panels where IFNs were measured.10) I am again confused.Regarding Supplementary Figure 1, the authors says: repeated passages in K18-ACE2……are the viral passages performed in cell culture or in mice???And also, why do they present now tree lineages of Betain panel B? There is a total lack of symmetry throughout the paper.11) Sorry, but I do not understand Supplemental Figure 2. What does VAT and MAF mean?How do you calculate the mutant frequency?12) There are previous examples on antigenic variation in the absence of selective pressures for other viruses such foot-and-mouth disease virus that can be cited: Diez et al. J. Gen. Virol. 70, 3281, 1989Domingo et al J. Gen. Virol. 74: 2039, 1993 Reviewer #2 (Remarks to the Author): RE: Willett et al. "SARS-CoV-2 evolution in the absence of selective immune pressures, results in antibody resistance, interferon suppression and phenotypic differences by lineage."SUMMARY: Willett et al. demonstrated that serial passages of SARS-CoV-2 in mice expressing human ACE2 receptors in a minimal to moderate selective pressure result in human-adapted SARS-CoV-2 lineages with the accumulation of genetic changes relevant to antibody resistance.
MAJOR COMMENTS This is a relatively complete genetic analysis of SAR-CoV-2 evolution in vivo in mouse models, with potentially important clinical implications.The study was well-designed and implemented.Some changes should be made prior to its acceptance for publication.
Concerning the selective pressure in this study, the authors believe that their work "did not constitute gain-of-function research as virus was unmodified and represented circulating variants with the study conducted without experimental selective pressures (Page 16)."This and other similar statements in the main text should be interpreted cautiously.The reviewer considers that serial passages of viruses actually constitute selective pressures for viruses that are potentially having gain-of-function characteristics.The authors need to revise their statements in the title and the main text.Also, the biosafety level (BSL-3) should be elevated to higher levels for processing all viruses-containing samples.
Table 1: There are many interesting and potentially important human interaction proteins (e.g., ZDHHC5 and HGOLGA7).However, these data are descriptive in nature without experimental evidence that supports their relevance in SAR-CoV-2 infectivity in this study.The authors may consider providing additional experiments or providing this table in Supplementary Information.

OTHER / MINOR COMMENTS:
Line 26: Could you specify Wuhan-like SARS-CoV-2?Which strain do you refer to?If possible, avoid using "Wuhan" throughout the text.
Line 39: Treatment resistance is not standard terminology.Consider changing to "therapeutic Resistance." Line 98: "… study used transgenic mice expressing the human ACE2 receptor."Please provide more descriptions about the transgenic expression ACE2 receptor in mouse lungs and other tissues.
Line 108: "….more infectious virus after 24h of infection versus Delta P0 virus (p = 0.02) (Figure 3)."Please change "versus" to "than." Line 124: need to briefly explain, to a broader readership, the K18-ACE mice and the rationale to use this model in the study.
Line 135-136: change "vaccinated subjects" to "vaccinated human subjects" Line 154: Give the full term for abbreviation(s) at its first appearance in the main text (e.g., VAF on Page 7 and TCID50 within line 289).Reference citations: In many cases, more citations are needed to reinforce the findings and statements.Please provide additional references if available.
Line 384: Provide a reference for the Benjamini-Hochberg p-value adjustment.
Reviewer #3 (Remarks to the Author): Review for the manuscript "SARS-CoV-2 evolution in the absence of selective immune pressures, results in antibody 2 resistance, interferon suppression and phenotypic differences by lineage" (COMMSBIO-23-1680-T, Communications Biology) July 8, 2023 SARS-CoV-2 is the best studied virus from the perspectives of molecular biology, epidemiology, immunology, chemistry and biothermodynamics.The manuscript "SARS-CoV-2 evolution in the absence of selective immune pressures, results in antibody 2 resistance, interferon suppression and phenotypic differences by lineage" represents a significant step in the research of time evolution of SARS-CoV-2.Except for the contribution to the research on viruses, it also represents a contribution to the research on viral evolution.
The manuscript analyzes especially well the evolution of SARS-CoV-2 from the perspectives of epidemiology, biology, immunology and molecular biology.However, it is possible to include into the analysis the perspective of biothermodynamics, which gives a mechanistic model for changes in infectivity and pathogenicity during evolution of SARS-CoV-2.This research is available in the literature Popovic, M., Martin, J. H., & Head, R. J. (2023).COVID infection in 4 steps: Thermodynamic considerations reveal how viral mucosal diffusion, target receptor affinity and furin cleavage act in concert to drive the nature and degree of infection in human COVID-19 disease.Heliyon, 9(6), e17174.https://doi.org/10.1016/j.heliyon.2023.e17174Popovic, M. (2023).SARS-CoV-2 strain wars continues: Chemical and thermodynamic characterization of live matter and biosynthesis of Omicron BN. 1, CH.1.1 and XBC variants. Microbial Risk Analysis, 24, 100260. https://doi.org/10.1016/j.mran.2023.100260 Popovic, M., Pantović Pavlović, M., & Pavlović, M. (2023).Ghosts of the past: Elemental composition, biosynthesis reactions and thermodynamic properties of Zeta P.2, Eta B. 1.525,Theta P.3,Kappa B.1.617.1,Iota B.1.526,Lambda C.37 and Mu B.1.621The mechanistic model and driving force for changes in infectivity and pathogenicity can give a response to the question of why during evolution there has been an increase in infectivity and maintenance or slight change in pathogenicity of the new variants of SARS-CoV-2, as well as suppression of earlier variants by the new variants.
In summary, the manuscript provides a significant contribution to the field.This reviewer believes that it deserves high priority for publication after a minor revision.

Figure 4 :
Figure 4: missing labels A, B, C, and D in this figure.